β-LEAF assays determine β-lactamase production and cefazolin activity in S. aureus clinical isolates. β-LEAF assays were performed with two ATCC S. aureus control strains (known β-lactamase producer #1 and non-producer #2) and 25 S. aureus clinical isolates, with cefazolin as a test antibiotic. The different bacterial isolates were incubated with β-LEAF (probe) alone and β-LEAF and cefazolin respectively, and fluorescence was monitored over 60 min. The y-axis represents the cleavage rate of β-LEAF (measured as fluorescence change rate – milliRFU/min) normalized by bacterial O.D. (optical density) at 600 nm. The black bars depict cleavage rate when β-LEAF alone is used, to show β-lactamase production. The white bars depict cleavage rate of probe when both the probe and cefazolin are included in the reactions. The horizontal line indicates a proposed cut-off value (upper limit of mean ± 3X Std. deviation for strain #2, β-LEAF probe reaction) to demarcate β-lactamase production. Where the black and white bars are significantly different, the antibiotic is predicted to be less active. Results are presented as the average of three independent experiments (each experiment contained samples in triplicates) and error bars represent the standard error for all isolates, except #2. For #2, the error bar is 3X standard deviation.